Composite

Part:BBa_K3278001

Designed by: Ailin Zhou   Group: iGEM19_ShanghaiTech_China   (2019-10-13)


pDawn with T7 promoter

pDawn is a blue light control system which can start the expression of target gene under the inducement of blue light. More information in BBa_K1075044 added by iGEM13_Bonn.

However, expression leakage and delay are the two main problems of pDawn system. Leakage refers to the expression of target protein without light. According to our measurement, samples strictly kept away from light still had significant expression of GFP after being cultured for one night. Therefore, we carefully studied the gene circuits of pdawn system [2] [3] and hypothesized that enhancing the strength of the promoter of YF1 may enhance the repression of protein expression in the dark, thus solving the leakage problem. In this part, we use the T7 promoter to substitute the lacIq promoter in front of YF1 and Fixk2 and compare its performance with the original pDawn-mEGFP component based on both the efficiency of mEGFP expression and the leakage level. To examine mEGFP expression dynamics, different illuminating duration was used in our experiment.

pDawn with T7 promoter is abbreviated as T7c below. pDawn with tac promoter is abbreviated as tac below.

[Go for the protocol of characterization:https://2019.igem.org/Team:ShanghaiTech_China/LightControl]

pDawn-T7c-0 hour
Fig1(a). pDawn-T7c-0 hour
pDawn-T7c-6 hour
Fig1(b). pDawn-T7c-6 hour
pDawn-T7c-12 hour
Fig1(c). pDawn-T7c-12 hour
pDawn-T7c-24 hour
Fig1(d). pDawn-T7c-24 hour
pDawn-T7c-30 hour
Fig1(e). pDawn-T7c-30 hour
pDawn-T7c-36 hour
Fig1(f). pDawn-T7c-36 hour
Figure 1: Representative images of T7c under different light illumination-durations. The groups labeled ‘light-on’ were under light induction, while controls labeled ‘light-off’ were kept in the dark wrapped with aluminum foil. pictures were taken with the same parameters as the Alexa Fluor 488 dye, with the same 1000-ms exposure time for all images. The Brightfield images were captured with 300-ms exposure time.


Result: For T7c, the absolute fluorescence intensity reached a relatively high level in a short time, namely reacting faster to light induction, despite the fact that the leakage level was not reduced.


Firstly, we quantified the absolute expression level of the T7c.

Figure 2 zal.png
Figure 2: Average Absolute F.I. line graphs of pDawn-mEGFP, T7c, tac, showing the absolute expression capacity


Result: A significant increase of the absolute expression level compared to the original pDawn-mEGFP can be observed directly from the graph in the first several timepoints, indicating T7c’s good property of rapid reaction with time.

T7c highlighted a significant increase of the absolute expression level compared to the original pDawn-mEGFP, reacting rapidly with a short time range. As for the leakage problem, we expect a combination of T7c with other low leakage modified components such as YF2 in order to weaken the leakage while obtaining a relatively high expression level.

To see more changes on pDawn, go to the websites below.
origin pDawn: https://parts.igem.org/Part:BBa_K1075044
pDawn with T7 promoter: https://parts.igem.org/Part:BBa_K3278001
pDawn with mtac promoter: https://parts.igem.org/Part:BBa_K3278002 (better)
pDawn with tac promoter: https://parts.igem.org/Part:BBa_K3278005
pDawn with YF2: https://parts.igem.org/Part:BBa_K3278006 (better)


References:
[1] From Dusk till Dawn: One-Plasmid Systems for Light-Regulated Gene Expression. Robert Ohlendorf1, Roee R. Vidavski, Avigdor Eldar, Keith Moffat, and Andreas Möglich. Journal of Molecular Biology, 2012, Vol.416, pp. 534-542.
[2] Design and Signaling Mechanism of Light-Regulated Histidine Kinases. Andreas Möglich, Rebecca A. Ayers and Keith Moffat. Biophysical Journal, 2009, Vol.96 (3), pp. 1433-1444.
[3] Oxygen-Regulated In Vitro Transcription of Rhizobium meliloti nifA and fixK Genes. JEAN-MARC REYRAT,' MICHEL DAVID,' CASIMIR BLONSKI,2 PIERRE BOISTARD,' AND JACQUES BATUT'*. Journal of Bacteriology, 1993, pp. 6867-6872.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2231
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 109
    Illegal NgoMIV site found at 241
    Illegal NgoMIV site found at 335
    Illegal NgoMIV site found at 628
    Illegal NgoMIV site found at 1122
    Illegal NgoMIV site found at 1140
    Illegal NgoMIV site found at 1230
    Illegal AgeI site found at 460
    Illegal AgeI site found at 1588
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1683
    Illegal BsaI.rc site found at 571


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